Maintenance of tissue adenine nucleotide (And=ATP+ADP+AMP) concentration is essential to support a broad range of cellular process. This is essentially true in skeletal muscle where ATP-dependent reactions can abruptly increase many fold during contractions. Processes that directly impact And metabolism involve the rate of energy expenditure (e.g., exercise intensity) and the activity of AMP deaminase (AMPD1), a tightly regulated enzyme. We have recently shown that a novel AMP- activated protein kinase (AMPK) phosphorylates purified AMPD1 in vitro; this induces a kinetic activation of AMPD1 that would be most significant at low physiological [AMP/f). This proposal evaluates the control of AMPD1 by enzyme phosphorylation/dephosphorylation, the involvement of AMPK, the role of AMP deaminase as 'gatekeeper' for And degradation, and the functional and metabolic consequences of absent or excessive AMP deamination in fast-twitch skeletal muscle in vivo. In vitro, in situ perfused, and in vivo preparations will be used to evaluate whether AMPD1 enzyme phosphorylation: contributes to AMP deaminase kinetic activation in vivo; occurs by activation of AMPK during contractions and via treatment with 5-aminoimadazone-4- carboxyamide ribonucleoside (AICAR) in vivo; and, is dephosphorylated by protein phosphatase 2A, leading to kinetic inactivation. We will evaluate the 'gatekeeper' role on AMPD1 on And degradation and recovery processes, by AMPD1 induced by AMPK. Further, we will assess whether absence of AMP deamination during contractions in vivo, established with a specific high-affinity inhibitor; AMPD1 gene deletion or adenylate kinase (AK1) gene deletion, impairs muscle function, and accelerates And degradation. These novel studies will contribute fundamental information to understand the control of AMP deamination in muscle, ' its impact on muscle function, and its role as a 'gatekeeper' for And metabolism in muscle.